Few subjects in medicine today arouse the interest of the scientific community and the lay community as does Alzheimer's disease (AD). AD has emerged as the most prevalent form of late-life mental failure in humans. AD is a common dementing brain disorder of the elderly. The key features of the disease include progressive memory impairment, loss of language and visuospatial skills, and behavior deficits. These changes in cognitive function are the result of degeneration of neurons in the cerebral cortex, hippocampus, basal forebrain, and other regions of the brain. Neuropathological analyses of postmortem Alzheimer's diseased brains consistently reveal the presence of large numbers of neurofibrillary tangles in degenerated neurons and neuritic plaques in the extracellular space and in the walls of the cerebral microvasculature. The neurofibrillary tangles are composed of bundles of paired helical filaments containing hyperphosphorylated tau protein (Lee & Trojanowski, 1992, Curr. Opin. Neurobiol. 2:653–656). The neuritic plaques consist of deposits of proteinaceous material surrounding an amyloid core (Selkoe, 1994, Annu. Rev. Neurosci. 17:489–517).
AD has been estimated to affect more than 4 million people in the United States alone and perhaps 17 to 25 million worldwide. Moreover, the number of sufferers is expected to grow as the population ages. The pathology of AD has been studied extensively for the last 20 years, but it was not until about 15 years ago that the first molecular handle in understanding this complex degenerative disease was obtained, when the protein sequence of the extracellular amyloid was determined.
The effort to decipher the mechanism of AD has attracted the interest of investigators from diverse biological disciplines, including biochemistry, cell biology, molecular genetics, neuroscience, and structural biology. The eclectic nature of research approaches to AD and the intensity of scientific interest in the problem have made it increasingly likely that AD will become a premier example of the successful application of biological chemistry to the identification of rational therapeutic targets in a major human disease. Much of the recent progress in elucidating the pathogenesis of AD has centered on the apparent role of the 40–42-residue amyloid-protein (Aβ) as a unifying pathological feature of the genetically diverse forms of this complex disorder.
AD is divided into 2 classes: Familial AD, (FAD) which has an early onset and is heritable, and “non-familial”, or sporadic AD (SAD), which has no identifiable cause. Although FAD is rare (less than 10% of all AD), the characteristic clinicopathological features—amyloid plaques, neurofibrillary tangles, synaptic and neuronal loss, and neurotransmitter deficits are apparently indistinguishable from the more common SAD.
The defining neuropathological characteristic of AD is the accumulation of insoluble proteinacious deposits, known as amyloid plaques, in the brains of those affected. The presence of these amyloid plaque deposits is the essential observation underpinning the amyloid hypothesis.
Evidence suggests that deposition of amyloid-β peptide (Aβ) plays a significant role in the development of amyloid plaques and the etiology of AD. For example, individuals with mutations in the gene encoding the β-amyloid precursor protein (APP) from which the Aβ protein is derived invariably develop Alzheimer's disease (Goate et al., 1991, Nature 353:844–846; Mullan et al., 1992, Nature Genet. 1:345–347; Murrell et al., 1991, Science 254:97–99; Van Broeckhoven, 1995, Eur. J. Neurol. 35:8–19). Likewise, autopsies have shown that amyloid plaques are found in the brains of virtually all Alzheimer's patients and that the degree of amyloid plaque deposition correlates with the degree of dementia (Cummings & Cotman, 1995, Lancet 326:1524–1587).
That increased expression and/or abnormal processing of APP is associated with the formation of amyloid plaques and cerebrovascular amyloid deposits, which are one of the major morphological hallmarks of AD has been corroborated from least two sources. The first is that transgenic mice which express altered APP genes exhibit neuritic plaques and age-dependent memory deficits (Games et al., 1995, Nature 373:523–525; Masliah et al., 1996, J. Neurosci. 16:5795–5811; Hsiao et al., 1996, Science 274:99–103).
The second body of evidence comes from study of patients suffering from Down's syndrome, who develop amyloid plaques and other symptoms of Alzheimer's disease at an early age (Mann & Esiri, 1989, J. Neurosci. 89:169–179). Because the APP gene is found on chromosome 21, it has been hypothesized that the increased gene dosage which results from the extra copy of this chromosome in Down's syndrome accounts for the early appearance of amyloid plaques (Kang et al., 1987, Nature 325:733–736; Tanzi et al., 1987, Science 235:880–884). Taken together with the evidence derived from cases of familial Alzheimer's disease, the current data suggest that genetic alterations which result in an increase in Aβ production can induce Alzheimer's disease. Accordingly, since Aβ deposition is an early and invariant event in Alzheimer's disease, it is believed that treatment which reduces production of Aβ will be useful in the treatment of this disease. Among the processes regulating APP metabolism, the proteolytic cleavage of APP into amyloidogenic or nonamyloidogenic fragments is of special interest.
The strongest evidence implicating Aβ in the pathogenesis of AD comes from the observation that Aβ peptides are toxic to neurons in culture and transgenic mice that overproduce Aβ in their brains show significant deposition of Aβ into amyloid plaques and significant neuronal toxicity (Yankner et al., 1989, Science 245:417–420; Frautschy et al., 1991, Proc. Natl. Acad. Sci. USA 88:8362–8366; Kowall et al., 1991, Proc. Natl. Acad. Sci. USA 88:7247–7251). This toxicity is enhanced if the peptides are “aged” (incubated from hours to days), a procedure that increases amyloid fibril formation. As well, injection of the insoluble, fibrillar form of Aβ into monkey brains results in the development of pathology (neuronal destruction, tau phosphorylation, microglial proliferation) that closely mimics Alzheimer's disease in humans (Geula et al., 1998, Nature Medicine 4:827–831). See Selkoe, 1994, J. Neuropathol. Exp. Neurol. 53:438–447 for a review of the evidence that amyloid plaques have a central role in Alzheimer's disease.
While abundant evidence suggests that extracellular accumulation and deposition of Aβ is a central event in the etiology of AD, recent studies have also proposed that increased intracellular accumulation of Aβ or amyloid containing C-terminal fragments may play a role in the pathophysiology of AD. For example, over-expression of APP harboring mutations which cause familial AD results in the increased intracellular accumulation of C100 in neuronal cultures and Aβ42 in HEK 293 cells. Aβ42 is the 42 amino acid long form of Aβ that is believed to be more efficacious at formed amyloid plaques than shorter forms of Aβ. Moreover, evidence suggests that intra- and extracellular Aβ are formed in distinct cellular pools in hippocampal neurons and that a common feature associated with two types of familial AD mutations in APP (“Swedish” and “London”) is an increased intracellular accumulation of Aβ42. Thus, based on these studies and earlier reports implicating extracellular Aβ accumulation in AD pathology, it appears that altered APP catabolism may be involved in disease progression.
APP is an ubiquitous membrane-spanning (type 1) glycoprotein that undergoes a variety of proteolytic processing events. (Selkoe, 1998, Trends Cell Biol. 8:447–453). APP is actually a family of peptides produced by alternative splicing from a single gene. Major forms of APP are known as APP695, APP751, and APP770, with the subscripts referring to the number of amino acids in each splice variant (Ponte et al., 1988, Nature 331:525–527; Tanzi et al., 1988, Nature 331:528–530; Kitaguchi et al., 1988, Nature 331:530–532). APP is expressed and constitutively catabolized in most cells.
APP has a short half-life and is metabolized rapidly down two pathways in all cells. The dominant catabolic pathway appears to be cleavage of APP within the Aβ sequence by α-secretase, resulting in the constitutive secretion of a soluble extracellular domain (sAPPα) and the appearance of a nonamyloidogenic intracellular fragment (approximately 9 kD), referred to as the constitutive carboxy-terminal fragment (cCTFα). cCTFα is a suitable substrate for cleavage by γ-secretase to yield the p3 fragment. This pathway appears to be widely conserved among species and present in many cell types (Weidemann et al., 1989, Cell 57:115–126; Oltersdorf et al., 1990, J. Biol. Chem. 265:4492–4497; and Esch et al., 1990, Science 248:1122–1124). In this pathway, processing of APP involves proteolytic cleavage at a site between residues Lys16 and Leu17 of the Aβ region while APP is still in the trans-Golgi secretory compartment (Kang et al., 1987, Nature 325:773–776). Since this cleavage occurs within the Aβ portion of APP, it precludes the formation of Aβ. sAPPα has neurotrophic and neuroprotective activities (Kuentzel et al., 1993, Biochem. J. 295:367–378).
In contrast to this non-amyloidogenic pathway involving α-secretase described above, proteolytic processing of APP by β-secretase exposes the N-terminus of Aβ, which after γ-secretase cleavage at the variable C-terminus, liberates Aβ. This Aβ-producing pathway involves cleavage of the Met671-Asp672 bond (numbered according to the 770 amino acid isoform) by β-secretase. The C-terminus is actually a heterogeneous collection of cleavage sites rather than a single site since γ-secretase activity occurs over a short stretch of APP amino acids rather than at a single peptide bond. In the amyloidogenic pathway, APP is cleaved by β-secretase to liberate sAPPβ and CTFβ, which CTFβ is then cleaved by γ-secretase to liberate the harmful Aβ peptide.
Of key importance in this Aβ-producing pathway is the position of the γ-secretase cleavage. If the γ-secretase cut is at residue 711–712, short Aβ (Aβ40) is the result; if it is cut after residue 713, long Aβ (Aβ42) is the result. Thus, the γ-secretase process is central to the production of Aβ peptide of 40 or 42 amino acids in length (Aβ40 and Aβ42, respectively). For a review that discusses APP and its processing, see Selkoe, 1998, Trends Cell. Biol. 8:447453; Selkoe, 1994, Ann. Rev. Cell Biol. 10:373–403. See also, Esch et al., 1994, Science 248:1122.
Aβ, the principal component of amyloid plaques, is a 39–43 amino acid peptide which is capable of forming β-pleated sheet aggregates. These aggregating fibrils are subsequently deposited in the brain parenchyma or in the cerebrovasculature of the Alzheimer's disease victim (Glenner et al., 1984, Biochem. Biophys. Res. Comm. 120:885–890; Masters et al., 1985, Proc. Natl. Acad. Sci. USA 82:4245–4249).
Reports show that soluble β-amyloid peptide is produced by healthy cells into culture media (Haass et al., 1992, Nature 359:322–325) and in human and animal CSF (Seubert et al., 1992, Nature 359:325–327).
Cleavage of APP can be detected in a number of convenient manners, including the detection of polypeptide or peptide fragments produced by proteolysis. Such fragments can be detected by any convenient means, such as by antibody binding. Another convenient method for detecting proteolytic cleavage is through the use of a chromogenic β-secretase substrate whereby cleavage of the substrate releases a chromogen, e.g., a colored or fluorescent, product.
Various groups have cloned and sequenced cDNA encoding a protein that is believed to be β-secretase (Vassar et al., 1999, Science 286:735–741; Hussain et al., 1999, Mol. Cell. Neurosci. 14:419–427; Yan et al., 1999, Nature 402:533–537; Sinha et al., 1999, Nature 402:537–540; Lin et al., 2000, Proc. Natl. Acad. Sci. USA 97:1456–1460). β-secretase has been called various names by these groups, e.g., BACE, Asp2, memapsin2.
Much interest has focused on the possibility of inhibiting the development of amyloid plaques as a means of preventing or ameliorating the symptoms of Alzheimer's disease. To that end, a promising strategy is to inhibit the activity of at least one of β- and γ-secretase, the two enzymes that together are responsible for producing Aβ. This strategy is attractive because, if the formation of amyloid plaques as a result of the deposition of Aβ is a cause of Alzheimer's disease, inhibiting the activity of one or both of the two secretases would intervene in the disease process at an early stage, before late-stage events such as inflammation or apoptosis occur. Such early stage intervention is expected to be particularly beneficial (see, e.g., Citron, 2000, Molecular Medicine Today 6:392–397).
Thus, it is believed that a drug that could interfere with β-amyloid plaque formation or toxicity may delay or halt the progression of Alzheimer's disease. At present, few suitable in vitro systems or methods exist for screening candidate drugs for the ability to inhibit or prevent the production of β-amyloid plaque. The scarcity of such screening methods may, at least in part, result from insufficient understanding of the pathogenic mechanism(s) which cause the conversion of amyloid precursor protein to the β-amyloid peptide, and ultimately to the amyloid plaque.
In view of the anticipated benefits of modulating APP catabolism as a treatment for diseases such as AD, compositions and methods for modulating APP catabolism in APP-containing cells which do not substantially alter the viability of those cells, have been desired and are addressed by the present invention.
For these reasons, it would be desirable to provide methods and systems for screening test compounds for the ability to inhibit or prevent the production of Aβ from APP. In particular, it would be desirable to base such methods and systems on a metabolic pathway which is involved in such conversion, where the test compound would be able to interrupt or interfere with the metabolic pathway which leads to conversion. In particular, initial methods should utilize in vitro systems rather than animal models, so that the methods are particularly suitable for initial screening of test compounds to identify suitable candidate drugs.